Abstract
BackgroundS-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering.ResultsA novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea.ConclusionsMutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure–function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH.
Highlights
S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12)
This enzyme is involved in the glutathionedependent formaldehyde detoxification pathway, which prevents the negative effects of formaldehyde by a series of enzymatic reactions involving formaldehyde dehydrogenase (FALDH, S-hydroxymethylglutathione dehydrogenase) and SFGH, which depend upon the spontaneous binding of glutathione to formaldehyde
A mutant strain of Paracoccus denitrificans for the SFGH gene was unable to grow on methanol or methylamine, which resulted in the formation of formaldehyde [6]
Summary
S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). Esterases hydrolyze ester bonds and produce cleaved acid and alcohol compounds They have low sequence identity and diverse substrate specificities as well as biological functions, most esterases adopt a common α/β hydrolase fold and contain a highly conserved serine–histidine–aspartic acid catalytic triad at the active site [1,2,3]. SFGH hydrolyzes S-formylglutathione into glutathione and formic acid and can be broadly considered an esterase, or a thioesterase. Esterase D, which was subsequently proven to be identical to SFGH, has been identified, characterized, and structurally determined This enzyme has attracted much attention as its polymorphisms are related to several diseases, including retinoblastoma and Wilson’s disease [7,8,9]. Bacterial SFGHs were identified in various species including Paracoccus denitrificans [6], Agrobacterium tumefaciens [15], and E. coli [5]
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